ABSTRACT Viral latency has emerged as the main barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill'-i.e. reactivation of latent virus using non- targeted small molecules. However, significant hurdles must be overcome for this approach to be successful. These include stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upon viral reactivation. Thus, an alternative strategy for targeting HIV-1 latency would make use of biomarkers to identify and selectively target latently infected cells in vivo without the need for reactivation. Additionally, these biomarkers would be invaluable for in vitro research given that patient-derived, native-state latently infected cells could now be purified for study. Unfortunatel however, such biomarkers do not currently exist. Here, we propose a multi-pronged, systems-biology based strategy to identify latency biomarkers in primary CD4+ T-cells. We propose to use a novel dual-fluorescence reporter HIV-1 genome to identify, quantify, and purify latently infected cells early post infection (<4 days), and without reactivation. Infection of primary CD4+ T-cells with this virus yields a reversible state of HIV latency in ~30% of infection events, which is a much higher frequency than many other currently used primary cell latency models. This model's unique set of characteristics is fundamental to our proposal to use a powerful combination of phenotyping tools to characterize the cells that become latent after HIV-1 infection of primary CD4+ T-cells. These tools will include: 1. CyTOF single-cell analysis of cell-surface markers, T-cell signaling networks, and T-cell effector function. 2. Quantitative mass spectrometry based identification of cell surface proteins enriched on latently infected cells in comparison to productively infected cells; 3. RNAseq and microRNAseq analysis of the cellular transcriptome of latently infected cells in comparison to productively infected cells. Most importantly, we will also screen our list of putative biomarkers for their predictive utility. Thiswill be done by validation not only in cells infected in vitro with the dual fluorescence virus, but als ex vivo in CD4+ T-cells isolated from HIV infected patients. We believe that this multi-pronged validation approach is vital to identifying bona fide biomarkers, and is likely to yield targets tht are invaluable for ex vivo validation of novel HIV-1 latency modulating therapeutics.